Recovery of plasmids in an aqueous two-phase system

ABSTRACT

The present invention is a method for the purification of plasmid DNA comprising to provide a composition comprising a first polymer having inverse solubility characteristics and a second polymer immiscible in the first polymer; contacting said solution with an aqueous solution comprising plasmid DNA; providing phase separation and isolating the aqueous phase; and increasing the temperature of the isolated phase to a temperature above the cloud point of the first polymer and below the temperature where plasmid DNA is degraded and subsequently isolating the aqueous phase so formed. The invention also encompasses a kit for purification of plasmid DNA as described above.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. §371 and claims priority tointernational patent application number PCT/SE2003/001016 filed Jun. 17,2003, published on Mar. 11, 2004 as WO 2004/020629 and also claimspriority to patent application number 0202552-6 filed in Sweden on Aug.27, 2002; the disclosures of which are incorporated herein by referencein their entireties.

TECHNICAL FIELD

The present invention relates to a method for the purification ofplasmid DNA in an aqueous two-phase system. The invention alsoencompasses a kit for purification of plasmid DNA from a cell lysate inan aqueous two-phase system. In addition, the present invention relatesto the use of certain polymers in a two-phase system for thepurification of plasmid DNA from a cell lysate.

BACKGROUND

One of the ways that genetic variability is maintained within apopulation is through recombination, a process involving the exchange ofgenetic information among different DNA molecules that results in areshuffling of genes. To provide recombination in the field of geneticengineering, a vector is usually used. The most commonly used vector isthe DNA plasmid, a small genetic element that permits microorganisms tostore additional genetic information.

Plasmids are useful elements in many biotechnological applications thesedays. For example, in the medical and diagnostic fields, geneticengineering of cells is performed using plasmids that carry a geneencoding a protein, which is not expressed in the native cell.

Another use of plasmids as vectors is in the field of gene therapy,which is expected to be one of the fastest growing areas in the nextdecade. Gene therapy is a therapeutic strategy where nucleic acids areintroduced to human cells to cure genetic defects e.g. cystic fibrosis.The first human gene therapy trials began in 1990, using an ex vivostrategy. In this approach, the patient cells are harvested andcultivated in the laboratory and then incubated with vectors tointroduce the therapeutic genes. Even though approaches for deliveringgenes based on in vivo gene therapy, in which the virus is directlyadministered to the patients, have been suggested more recently as analternative, the plasmid retains its importance in gene therapy.

Thus, the increased use of such biotechnological applications results ina need for large quantities of plasmid DNA. To this end, an efficientlarge-scale purification process, which can meet specifications inpurity and quantitation, is required. Today, many purification methodsare available for smaller molecules of sizes of about 10 nm, such asproteins. However, for the larger DNA plasmids, which are of sizes of100 nm and above, much fewer purification methods are available.

Conventionally, the production of plasmid DNA involves fermentation,primary purification and high-resolution separation. Recently manymethods have been suggested involving use of chromatography as themethod for purification of plasmid DNA. However, the use ofchromatography as a single purification technique alone for plasmid DNAinvolves several drawbacks, such as a slow diffusion, low capacity ofthe matrices, shearing of large plasmids and recovery of the plasmid inhigh salt concentration. Therefore, there is a need of a primarypurification step before chromatography.

Use of a two-phase system has been suggested for purification of plasmidDNA. Aqueous two-phase systems are extremely mild and have shown astrong potential for use as a primary recovery step for plasmidpurification. Plasmid DNA is today often produced in Escherichia coliand involves an alkaline lysis step for release of plasmid DNA from thebacterial cells. Several contaminants such as RNA, genomic DNA,proteins, cells and cells debris are released in the alkaline lysisstep. Ribeiro et al. (S. C. Ribeiro et al: Isolation of Plasmid DNA fromCell Lysates by Aqueous Two-Phase Systems, 2002 Wiley Periodicals) hasshown that plasmid DNA can be isolated in aqueous two-phase systemsconsisting of polyethylene glycol (PEG) and a salt. PEG is a linearpolymer of ethylene oxide groups. The polymer is soluble in water, andat a certain salt concentration a two-phase system consisting of PEG andsalt can be achieved. The PEG polymer can be removed by filtration ordialysis, which however often decreases the yield. Another drawback withthis method is that PEG is a relatively expensive chemical, which willbe of importance in large-scale processes. Furthermore, plasmid DNAresulting from this method will be present in an environment of highsalt concentration, which is a disadvantage in applications such as genetherapy.

An alternative two-phase system differs from the PEG/salt systems, inthe sense that the system is created by temperature-induced phaseseparation. More specifically, this means that a thermoseparatingpolymer is used, which polymer solution will separate into two phaseswhen its temperature is increased to a point above its cloud point (CP).The above discussed PEG polymer can in fact be used as athermoseparating polymer, but its high cloud point, which is 111.7° C.at a 10% solution in water, renders PEG systems highly unsuitable forseparation of delicate biological materials. Thermoseparating two-phasesystems have been suggested for partitioning of some proteins, such asenzymes, and for a water-soluble steroid.

More specifically, Harris et al. (P. A. Harris et al: Enzymepurification using temperature-induced phase formation, Bioseparation 2:237-246, 1991) disclose the purification of the enzyme3-phosphoglycerate kinase and hexokinase from a cell homogenate ofbaker's yeast in an aqueous two-phase system. The system used comprisesa random copolymer of ethylene oxide (EO) and propylene oxide (PO) knownas UCON 50-HB-5110 and dextran or hydroxypropyl starch. The EO-POcopolymer has a cloud point that is much lower than that of PEG, namely50° C.

Further, Alred et al (Patricia A. Alred et al: Partitioning ofectdysteroids using temperature-induced phase separation, Journal ofChromatography, 628 (1993) 205-214) discloses a study of thepartitioning of the ectdysteroids α-ecdysone and β-ecdysone in anaqueous two-phase system by thermoseparation. The system comprises thesame components as the above-mentioned, namely the ethyleneoxide-propylene oxide random copolymer UCON 50-HB-5100 and dextran. Dueto the high levels of ecdysteroids recovered, 73.6% and 85.6%,respectively, such a system is suggested as an analytical or apreparative technique for ecdysteroids.

Finally, Persson et al (Persson et al: Purification of recombinantapolipoprotein A-1 expressed in Escherichia coli using aqueous two-phaseextraction followed by temperature-induced phase separation, Journal ofChromatography B, 711 (1998) 97-109) describe a method of purificationof recombinant apolipoprotein A1 in aqueous two-phase systems comprisingethylene oxide-propylene oxide random copolymers and hydroxypropylstarch. The polymer system is thermoseparating in the sense that itseparates into one water-rich and one polymer-rich phase when heatedabove a critical point. It was shown that apolipoprotein could bepartitioned to the top EO-PO copolymer phase while contaminatingproteins and DNA was partitioned to the bottom phase.

In summary, there is still a need of improved methods for thepurification of plasmid DNA from cell lysates.

SUMMARY OF THE PRESENT INVENTION

The object of the present invention is to provide a method for thepurification of plasmid DNA, which avoids one or more of theabove-discussed drawbacks of the prior art. This can be achieved by themethod as defined in the appended claim 1.

Thus, one object of the invention is to provide a method, which isuseful as a primary step in plasmid purification.

A specific object is to provide a method for the purification of plasmidDNA in an aqueous two-phase system, which results in a product whereinthe salt concentration is lowered as compared to the prior art method.

Another object of the invention is to provide a method, which is capableof separating plasmid DNA from RNA and other impurities, such as proteinand genomic DNA, in a cell lysate.

A further object of the invention is to provide a kit useful forseparation of plasmid DNA from a cell lysate in an aqueous two-phasesystem comprising a thermoseparating polymer.

Other aspects and advantages of the present invention will appear fromthe detailed description of the present invention and the experimentalpart below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic picture of the purification of plasmid DNA in athermoseparating system comprising a first and a second polymeraccording to the invention. Plasmid DNA is partitioned to the top phasein the first system, the top-phase is then isolated and heated. A newtwo-phase system is obtained, wherein the plasmid DNA is recovered inthe water phase.

FIG. 2 shows an agarose gel analysis of top and bottom phases from athermoseparating system comprising a first and a second polymer and 50mM Na₂HPO₄ buffer system according to the invention.

FIG. 3 shows an agarose gel analysis of top and bottom phases from athermoseparating system comprising a first and a second polymer and 50mM Na₂HPO₄ buffer system according to the invention, wherein thedilution of the thermoseparated top phase has been varied.

FIG. 4 shows an agarose gel analysis of thermoseparating systemcomprising a first and a second polymer and 50 mM Na₂HPO₄ buffer systemaccording to the invention, wherein the first polymer/second polymerconcentration has been varied in the primary system.

FIG. 5 shows agarose gel electrophoresis (0.8% w/v) of top and bottomphases from extraction of a diafiltrated lysate in an aqueous two-phasesystem as described in example 2 below.

FIG. 6 shows agarose gel electrophoresis (0.8% w/v) of top and bottomphases from extraction of an ultrafiltrated lysate in an aqueoustwo-phase system as described in example 3 below.

DEFINITIONS

The term “a biological solution” embraces any solution that comprisesplasmid DNA, usually together with undesired contaminating componentssuch as cell debris, proteins, RNA etc.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is a method for the purificationof plasmid DNA in an aqueous two-phase system, comprising the steps of

-   (a) providing a composition comprising a first polymer that exhibits    inverse solubility characteristics, a second polymer that is    immiscible in the first polymer and optionally a salt;-   (b) contacting said solution with an aqueous solution comprising    plasmid DNA;-   (c) providing a phase separation and subsequently isolating the    aqueous phase;-   (d) increasing the temperature of the isolated phase to a    temperature above the cloud point of the first polymer and below the    temperature where plasmid DNA is degraded and subsequently isolating    the aqueous phase so formed; and optionally-   (e) a chromatography step to recover the plasmid DNA from the    isolated top phase.

Thus, the first and the second polymers used in the present method areimmiscible. More specifically, one polymer should be essentiallyhydrophilic and the other should be more hydrophobic but stillwater-soluble, i.e. amphiphilic. As the skilled person in this fieldwill understand, the concentrations of the first and second polymershould be high enough to bring about a phase separation into at leasttwo phases.

Thus, the first polymer is amphiphilic, water-soluble and capable tointeract with plasmid DNA. That way, the plasmid DNA will be extractedinto the more hydrophobic phase and thereby separated from morehydrophilic contaminants such as proteins, cells, cell debris etc. Theterm “inverse solubility” means that the solubility of the polymervaries inversely with the solution temperature, and more specifically,that the solubility of the polymer decreases with increasing solutiontemperature. Inverse solubility is therefore directly opposed to thetemperature effect exhibited by most solutes. In the present context,the term “thermoseparating” is also used to denote the first polymer.Quite surprisingly, the present inventors have also shown that plasmidDNA can be separated from genomic DNA and RNA using the method accordingto the invention. In fact, the present invention shows for the firsttime that plasmid DNA can be obtained in a water solution free frompolymer after extraction in an aqueous two-phase system.

Examples of suitable polymers useful as the first polymer that exhibitsinverse solubility can e.g. be found in I. Y. Galaev et al., EnzymeMicrob. Tech., vol 15 (1993), pp. 354-366. More specifically, in oneembodiment, the first polymer is selected from the group that consistsof polyalkylene glycols, such as hydrophobically modified polyalkyleneglycols, poly(oxyalkylene)polymers, poly(oxyalkylene)copolymers, such ashydrophobically modified poly(oxyalkylene)copolymers, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinylmethylether, alkoxylated surfactants, alkoxylated starches, alkoxylatedcellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyethers,and poly N-isopropylacrylamide and copolymers thereof. Derivatives andmixtures of the above-mentioned examples are also included within thescope of the present invention. Also, WO 98/11140 (Pharmacia & UpjohnAB) provides a more detailed description of suitable thermoseparatingpolymers useful in the present method. In an advantageous embodiment ofthe present method, the first polymer is a copolymer comprised ofethylene oxide and propylene oxide. In a preferred embodiment, thecopolymer is comprised of about 50% of ethylene oxide and about 50% ofpropylene oxide. Polymers of this kind are commercially available, suchas Breox PAG 50 A 1000 (available from Laporte Performance Polymers,Southampton, U.K).

The concentration of the first polymer in the aqueous solution can be inthe range of from about 0.5% up to about 30% by weight of the totalweight of the aqueous solution, preferably within the range of about3-20% by weight, more preferably within the range of about 4-15% byweight and most preferably about 4.5% by weight.

The second polymer is as mentioned above less hydrophobic than the firstpolymer and water-soluble. The molecular weight of the second,hydrophilic polymer may be in the range of from about 3,000-5,000,000,such as 10,000-5000,000 and preferably in the range from 40,000-500,000.In an advantageous embodiment of the present method, the second polymeris selected from the group that consists of hydroxyalkyl cellulose,hydroxyalkyl starches, starch, dextran, pullulan and derivatives andmixtures thereof. Polymers of this kind are also commercially available,such as e.g. Dextran 500 T (Amersham Biosciences AB, Uppsala, Sweden).The concentration of the second polymer in the aqueous solution can bein the range of from about 1% up to about 30% by weight of the totalweight of the aqueous solution, preferably within the range of about3-20% by weight, more preferably within the range of about 4-15% byweight and most preferably about 4.5% by weight.

The present invention also encompasses the use of three or morepolymers. Such multiphase separation can be designed by the skilledperson in this field by selecting suitable combinations of three or morepolymers and sufficiently high concentrations thereof when contactedwith the aqueous solution.

As the skilled person in this field will realise, the concentration ofplasmid DNA in the aqueous solution, i.e. the primary system, willdepend on its origin. For example, if the aqueous solution is a celllysate, the concentration of plasmid DNA in the starting material willdepend on the production host. The concentration of plasmid DNA in theprimary system is typically between 0.1 μg/ml-500 μg/ml.

As appears from the above, in one embodiment of the method according tothe invention, the weight ratio of the amounts of first polymer:secondpolymer is about 1:1. In an advantageous embodiment of the presentmethod, the amount of the first polymer is 4.5% (w/w) and the amount ofthe second polymer is 4.5% (w/w) of the composition provided in step(a). This embodiment is especially advantageous if a more extremepartitioning to the top phase is desired.

The partitioning of plasmid DNA to the hydrophobic phase can be enhancedby adding a compound with a hydrophobic cation or, alternatively, ahydrophilic anion, to the aqueous solution. Suitable compounds includeinorganic salts containing cations such as straight or branchedtrimethyl ammonium, triethyl ammonium, tripropyl ammonium, tributylammonium, tetramethyl ammonium, tetraethyl ammonium, tetrapropylammonium and tetrabutyl ammonium, and anions such as phosphates,sulphate, nitrate, chloride and hydrogen carbonate. Specific examplesare triethyl ammonium sulphate and sodium phosphate. The concentrationof a compound with a hydrophobic cation or hydrophilic anion should beselected to give an enhanced partitioning effect, while at the same timeavoiding precipitation of plasmid DNA.

The partitioning of molecules between the phases of two-phase systems isdescribed by the partitioning coefficient K, which is defined asK=C _(T) /C _(B)  (1)wherein

-   -   C_(T)=the concentration in the top phase of the molecule of        interest    -   C_(B)=the concentration in the bottom phase of the molecule of        interest.

The partitioning of molecules between the phases of the two-phasesystems can be shown in phase diagrams, wherein the borderline betweenone and two phases is called the binodial curve. The polymerconcentration of the two phases in equilibrium with each other aredescribed by tie lines in the phase diagram. Increase of the polymerconcentration, i.e. increase of the tie line length, leads, to moreextreme partitioning in two-phase systems (see G. Johansson, Methods inEnzymology, vol. 228 (1994) pp. 28-42). The skilled person in this fieldcan easily perform experiments for arriving at conditions for suitablepartitioning between the two phases.

In one embodiment of the present method, the aqueous solution thatcomprises plasmid DNA is a cell lysate, which method comprises a stepfor desalting the cell lysate before step (b).

After the contact of step (b), the plasmid DNA is partitioned to thehydrophobic phase. As mentioned above, this partitioning can be enhancedby adding a compound such as a salt. As is well known, addition of saltwill also decrease the cloud temperature of the first polymer, andaccordingly the phase separation provided in step (d) could use a lowertemperature in that case. In the most convenient embodiment of thepresent method, the mixing according to step (b) and the partitioningaccording to step (c) are performed at room temperature. This firstphase separation results in a top phase comprising the first polymer andplasmid DNA in water, while the lower phase contaminating RNA andproteins in the second polymer and water.

In step (c), the top phase comprising the plasmid DNA is isolated in aseparate container. In this second phase separation of the presentmethod, the isolated phase is heated to a temperature above the cloudpoint of the thermoseparating polymer, which as mentioned above willdepend on the salt concentration in the sample. However, in a preferredembodiment, the temperature is increased, preferably in a water bath, toa temperature above about 40° C., such as about 50° C. The heatingresults in a phase separation, wherein the top phase comprises theplasmid DNA and the lower phase comprises the polymer. Thus, the plasmidDNA is obtained in a water phase which is essentially free of polymer,since the polymer content in the top phase has been shown to compriseless than 1% polymer. In addition, the salt concentration in thesolution wherein the plasmid DNA is obtained can be much lower than inthe suggested prior art method wherein plasmid DNA is salted out.

Accordingly, the product obtained from the method according to theinvention will be more advantageous than the prior art products forapplications wherein a high degree of purity is a requirement, such asfor gene therapy. Compared to other methods, such as a series ofchromatographic steps, aqueous two-phase systems are known to be easy toscale up, since the partitioning is essentially independent of the sizeof the system.

A last embodiment of this first aspect is a method for purification ofplasmid DNA from a lysate, which comprises a first step of desalting thelysate, a second step for recovery which comprises the method describedabove, and a last step of chromatography for final purification of theplasmid DNA. The desalting can be performed by any suitable method. Inone embodiment, the desalting is performed by a method selected from thegroup that consists of gel filtration, diafiltration andultrafiltration.

A second aspect of the present invention is a composition for extractionof plasmid DNA in an aqueous two-phase system, which compositioncomprises a first polymer that exhibits inverse solubilitycharacteristics at temperatures below about 60° C., a second polymerthat is immiscible in the first polymer and optionally a salt. The firstand the second polymers are as discussed above in relation to the methodaccording to the invention.

In one embodiment of the present composition, the amount of the firstpolymer is 4.5% (w/w) and the amount of the second polymer is 4.5%(w/w).

In a preferred embodiment, the present composition is for separationusing a method according to the invention.

A third aspect of the present invention is a kit for purification ofplasmid DNA from a cell lysate in an aqueous two-phase system, which kitcomprises a first polymer that exhibits inverse solubilitycharacteristics at temperatures below about 60° C., a second polymerthat is immiscible in the first polymer and optionally a salt in onecompartment as well as written instructions for the use thereof.

The first polymer is as discussed above. In an advantageous embodimentof the present kit, the first polymer is comprised of ethylene oxide andpropylene oxide. In an advantageous embodiment, said copolymer iscomprised of about 50% of ethylene oxide and about 50% of propyleneoxide. Such copolymers are as discussed in detail above.

In an advantageous embodiment of the present kit, the second polymer isselected from the group that consists of hydroxyalkyl cellulose,hydroxyalkyl starches, starch, dextran, pullulan and derivatives andmixtures thereof, as discussed in more detail above in relation to themethod according to the invention.

In a specific embodiment of the present kit, the weight ratio of theamounts of first polymer:second polymer is about 1:1.

In a preferred embodiment of the kit, the amount of first polymer is4.5% (w/w) and the amount of second polymer is 4.5% (w/w).

In the preferred embodiment, the present kit is for purification of acell lysate that has been desalted before being mixed with an aqueoussolution that comprises plasmid DNA.

In an advantageous embodiment the present kit is for use in a methodaccording to the present invention.

A fourth aspect of the present invention is the use of a polymer thatexhibits inverse solubility characteristics at temperatures below about60° C. in an aqueous two-phase system for the purification of plasmidDNA from a cell lysate.

The first polymer is as discussed above. In an advantageous embodiment,the polymer is a copolymer of ethylene oxide and propylene oxide. In thepreferred embodiment, the copolymer is comprised of about 50% ofethylene oxide and about 50% of propylene oxide.

In order to provide a two-phase system, the polymer is used togetherwith a second polymer that is immiscible therein. In the preferredembodiment, the second polymer is selected from the group that consistsof hydroxyalkyl cellulose, hydroxyalkyl starches, starch, dextran,pullulan and derivatives and mixtures thereof. In the most preferredembodiment, the second polymer is dextran. However, the second polymercan be as discussed above in relation to the method according to theinvention.

Finally, the present invention also encompasses the use of a polymerselected from the group that consists of hydroxyalkyl cellulose,hydroxyalkyl starches, starch, dextran, pullulan and derivatives andmixtures thereof in an aqueous two-phase system for the purification ofplasmid DNA from a cell lysate, wherein the purification is obtained bytemperature-induced phase separation.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic picture of the purification of plasmid DNA insystem according to the invention, wherein the thermoseparating polymeris EO₅₀PO₅₀ and the second polymer is Dextran T 500 (AmershamBiosciences AB, Uppsala, Sweden). The different forms of plasmid DNA arepartitioned to the top phase in the first system. The top phase is thenisolated in a new container and heated to a temperature above its cloudpoint (CP). A new two-phase system is obtained where the plasmid DNA isrecovered in the water phase free from polymer.

FIG. 2 shows an agarose gel analysis of top and bottom phases from athermoseparating EO₅₀PO₅₀/Dextran T 500 and 50 mM Na₂HPO₄ according tothe invention. A volume of 12 μl sample was added to each lane. Theagarose gel was stained with ethidium bromide. More specifically, lane 1is a blank;

lane 2 is a molecular weight marker; lane 3 is a desalted alkalinelysate; lane 4 is the bottom phase from thermoseparated system; lane 5is the bottom phase from thermoseparated system; lane 6 is the bottomphase primary system; lane 7 is the bottom phase primary system; lane 8is a thermoseparated top phase; lane 9 is a thermoseparated top phase;lane 10 is the top phase primary system; lane 11 is the top phaseprimary system; and lane 12 is a molecular weight marker. Here, the term“primary system” refers to the first phase separation provided accordingto step (c) of the present method, while the term “thermoseparatedsystem” refers to the phase separation provided according to step (d) ofthe present method.

FIG. 3 shows an agarose gel analysis of top and bottom phases from athermoseparating EO₅₀PO₅₀/Dextran T 500 and 50 mM Na₂HPO₄ according tothe invention. The agarose gel is stained with SYBR green. A volume of12 μl was added to each lane. More specifically, lane 1 is athermoseparated top phase 5× diluted; lane 2 is a thermoseparated topphase 5× diluted; lane 3 is a thermoseparated top phase 2× diluted; lane4 is a thermoseparated top phase 2× diluted; lane 5 is a thermoseparatedtop phase 1× diluted; lane 6 is a thermoseparated top phase 1× diluted;lane 7 is a top phase primary system 5× diluted; lane 8 is a top phaseprimary system 5× diluted; lane 9 is a top phase primary system 2×diluted; lane 10 is a top phase primary system 2× diluted; lane 11 is atop phase primary system 1× diluted; lane 12 is a top phase primarysystem 1× diluted; lane 13 is a bottom phase from thermoseparation 5×diluted; lane 14 is a bottom phase from thermoseparation 2× diluted;lane 15 is a bottom phase from thermoseparation 1× diluted; lane 16 is abottom phase primary system 5× diluted; lane 17 is a bottom phaseprimary system 5× diluted; lane 18 is a bottom phase primary system 2×diluted; lane 19 is a bottom phase primary system 2× diluted; lane 20 isa bottom phase primary system 1× diluted; lane 21 is a bottom phaseprimary system 1× diluted; lane 22 is a desalted alkaline lysate 5×diluted; lane 23 is a desalted alkaline lysate 2× diluted; and lane 24is a desalted alkaline lysate 1× diluted.

FIG. 4 shows an agarose gel analysis of thermoseparatingEO₅₀PO₅₀/Dextran T 500 and 50 mM Na₂HPO₄ according to the invention. Thesystems are composed of different polymer concentrations:

4/6=4% (w/w) E₅₀PO₅₀/6% (w/w) Dextran T 500,

3/7=3% (w/w) E₅₀PO₅₀/7% (w/w) Dextran T 500,

3/8=3% (w/w) EO₅₀PO₅₀/8% (w/w) Dextran T 500,

2.5/9=2.5% (w/w) EO₅₀PO₅₀/9% (w/w) Dextran T 500.

All systems contain 50 mM 50 mM Na₂HPO₄. A volume of 12 μl sample isadded to each lane. The agarose gel is stained with ethidium bromide.

More specifically, in FIG. 4, lane 1 is a blank; lane 2 is a molecularweight marker; lane 3 is a desalted alkaline lysate; lane 4 is thebottom phase from thermoseparation in a 3/7 system; lane 5 is the bottomphase from the primary step in a 3/7 system; lane 6 is thethermoseparated top phase in a 3/7 system; lane 7 is the top phase fromthe primary system in a 3/7 system; lane 8 is the bottom phase from thethermoseparation in a 4/6 system; lane 9 is the bottom phase from theprimary system in a 4/6 system; lane 10 is the thermoseparated top phasein a 4/6 system; lane 11 is the top phase from the primary system in a4/6 system; lane 12 is a molecular weight marker; lane 13 is a blank;lane 14 is a blank; lane 15 is a molecular weight marker; lane 16 is thebottom phase from thermoseparation in a 2.5/9 system; lane 17 is thebottom phase from a primary system in a 2.5/9 system; lane 18 is thethermoseparated top phase in a 2.5/9 system; lane 19 is the top phasefrom a primary system in a 2.5/9 system; lane 20 is the bottom phasefrom the thermoseparation in a 3/8 system; lane 21 is the bottom phasefrom a primary system in a 3/8 system; lane 22 is the thermoseparatedtop phase in a 3/8 system; lane 23 is the top phase from a primarysystem in a 3/8 system; and lane 24 is a molecular weight marker.

FIG. 5 shows agarose gel electrophoresis (0.8% w/v) of top and bottomphases from extraction of a diafiltrated lysate in an aqueous two-phasesystem as described in example 2 below. More specifically, in FIG. 5,lane 1 is the thermoseparated bottom phase; lane 2 is thethermoseparated top-phase; lane 4 is the bottom phase (primary system);lane 5 is the top phase (primary system); lane 7 is the diafiltratedlysate (starting material); and lane 9 is the Mw marker.

FIG. 6 shows agarose gel electrophoresis (0.8% w/v) of top and bottomphases from extraction of an ultrafiltrated lysate in an aqueoustwo-phase system as described in example 3 below. More specifically, inFIG. 6, lane 1 is the ultrafiltrated lysate (starting material); lane 2is the top phase in primary system; lane 3 is the thermoseparated topphase; lane 4 is the thermoseparated bottom phase; and lane 5 is the Mwmarker.

EXPERIMENTAL PART

The following examples are provided for illustrative purposes only andare not to be construed as limiting the scope of the present inventionas defined by the appended claims. All references given below andelsewhere in the present application are hereby included by reference.

Material and Methods

Chemicals

The polymer Breox PAG 50 A 1000 (EO50PO50) (Mr 3900) was obtained fromLaporte Performance Chemicals (Southampton, U.K.) and Dextran T 500,with a molecular weight of 500,000, is available from AmershamBiosciences AB (Uppsala, Sweden). Na₂HPO₄ was obtained from MerckEurolab. A high copy number plasmid pUC 19 (2.686 kb) with a JV4 insert(3.433 kb) was a gift from Amersham Biosciences. The plasmid is referredto as pJV4 later on in the text (Vasi 1999).

Cultivation

The plasmid pJV4 was cultivated in E. coli strain TG1α over night in 2.0L baffled shake flasks using 500 ml Lauria broth (10 g/L NaCl, 10 g/LTryptone and 5 g/L Yeast extract). The ampicillin concentration in thecultivations was 100 μg/ml and grown overnight at 37° C. at 250 rpm.

Alkaline Lysis

A modified alkaline lysis method was used. A 500 ml overnight cellculture was harvested by centrifugation at 9000 rpm in a Sorvall SLA3000-rotor for 10 min at 4° C. The supernatant is carefully removed and5 g of the bacterial pellet was resuspended by vortexing in 36 mLsuspension buffer consisting of 61 mm glucose, 10 mM Tris, 50 mM EDTA(pH 8). After the cells are completely resuspended, 78 mL of lysisbuffer P2 (0.2 M NaOH, 1% SDS) was added while stirring gently with amagnetic stirrer. The mixture was incubated for 10 min at roomtemperature while stirring, assuring a complete mixture (1 phase) isachieved. A volume of 58.6 mL of ice cold neutralisation buffer (5 Mpotassium acetate, pH 5.5) was added to the lysate. The solution waskept in an ice-bath on a magnetic stirrer for at least 20 min. A whiteprecipitate was formed containing genomic DNA, proteins and cell debris.The precipitate was then removed by centrifugation in an SS-34-rotor at4° C. for 30 minutes at 10 000 rpm. The supernatant is then carefullyremoved to a fresh tube and stored in the refrigerator.

Example 1 Separation of Plasmid pJV4 from an E. coli Lysate, Desaltingby Gel Filtration

The alkaline lysate was desalted by gel filtration on a Sephadex™ G-25(Amersham Biosciences AB, Uppsala, Sweden) matrix. More specifically,the Sephadex beads were packed in a XK 50/30 column (AmershamBiosciences AB, Uppsala, Sweden), giving a total bed volume of 225 ml.The column was integrated to an ÄKTA™ explorer 10 system (AmershamBiosciences AB, Uppsala, Sweden) and equilibrated with the mobile phase(5 mM sodium phosphate buffer). Samples to be desalted (50-100 ml) werepumped into the column by the sample pump P-950 at a flow rate of 5ml/min. The eluate from the column was monitored by UV absorbance andconductivity, which ensured an appropriate fractionation of nucleic acidcontaining eluate in 5 mM sodium phosphate buffer.

Aqueous Two-Phase Systems

Systems of a total weight of 10 g, containing 4.5% (w/w) Dextran T 500and 4.5% (w/w) EO₅₀PO₅₀ were made up by weighing appropriate amounts ofa 25% stock solution of dextran and 100% EO₅₀PO₅₀ stock solution in 10mL graduated test tubes. The buffer salts used were 50 mM Na₂HPO₄ andwas added to the system from a 1 M stock solution. The clarified anddesalted alkaline lysate was added to a final weight of 10 g. Thesystems were mixed carefully until all polymers were dissolved and thephases were then separated by centrifugation (1600 g, 10 minutes) atroom temperature. The volume of the top and bottom phase was determined.The phases were separated and isolated in new containers. The top phasewas placed in a water bath at 55° C. for three minutes and thencentrifuged for two minutes to obtain one water phase and oneconcentrated polymer phase.

Agarose Gel Electrophoresis

The top and bottom phases from the phase system were analysed on anagarose gel. The agarose gel was run in a Hoefer™ HE 33 mini submarineelectrophoresis unit from Amersham Biosciences AB. A 0.8% (w/v) agarose(Duchefa, Netherlands) gel containing 15 μg/ml ethidium bromide (QuantumBiotechnologies, USA) was run for 90 V and 30 min. The gel was analysedand photographed under UV light.

Results Example 1 Polymer Concentration and Salt Effects

The partitioning of desalted alkaline lysate containing the plasmid DNAin an aqueous two-phase system can be influenced by different factorssuch as polymer concentration and salt composition. The object was toobtain a one sided partitioning of the plasmid to the EO₅₀PO₅₀ phase inthe primary aqueous two-phase system (FIG. 1.). By altering theconcentration of the polymers in a two-phase system, plasmid DNA can bemore extremely partitioned to the top phase. Partitioning of plasmid DNAin desalted alkaline lysate in two-phase systems composed of differentconcentrations of EO₅₀PO₅₀ and Dextran T 500 was studied. By decreasingthe polymer concentration in both phases a more extreme partitioning tothe top phase was achieved (FIG. 2.). Qualitative analysis on an agarosegel electrophoresis shows that by decreasing the polymer concentrationfrom 7% (w/w) Dextran T 500/7% (w/w) EO₅₀PO₅₀ to 4.5% (w/w) Dextran T500 and 4.5% (w/w) EO₅₀PO₅₀, plasmid DNA can be partitioned to the topphase. It has earlier been described in the literature (Albertson, 1986)that DNA can effectively be transferred between phases by addition of asuitable salt. To achieve a dominating effect of the salt in a two-phasesystem, the concentration of the salt must be at least ten times higherthen the buffer. The addition of a salt to a two-phase system forces theanion and the cation to partition together between the two differentpolymer phases and this will generate an electrical potential betweenthe phases. The HPO₄ ²⁻ anion has affinity for the dextran phase. Thiswill create an electrochemical driving force in the system. If anegatively charged substance e.g. plasmid DNA is added to this system,the DNA will partition to the top phase (FIG. 2).

Partitioning of Plasmid DNA in a Thermoseparating System

The lysate was partitioned in systems containing 4.5% (w/w) Dextran T500, 4.5% (w/w) EO₅₀PO₅₀ and 50 mM Na₂HPO₄. The systems were preparedaccording to the section “Material and methods” above. The systems weremixed and after phase separation the phases were separated and isolatedin separate containers. The top phase was put in a water bath at 55° C.The system phase separated into two new phases, one water phase and onedense polymer phase. The thermoseparating step was utilised forseparating the target plasmid from the EO₅₀PO₅₀ polymer, and isolatingthe plasmid in a water phase with low (<1%) polymer solution. The phaseswere analysed on an agarose gel electrophoresis stained with ethidiumbromide (FIG. 2.) From the agarose gel electrophoresis it can be seenthat all forms of plasmid DNA are partitioned to the top phase in thefirst step and in the thermoseparating step the plasmid DNA isexclusively partitioned to the top water phase. From the agarose gelelectrophoresis (FIG. 2.) it can also be seen that contaminating RNA isto a high degree partitioned to the bottom phase in the primary system.By staining the agarose gel with SYBR green (Molecular Probes) a 25times higher sensitivity in detection of DNA can be achieved compared tostaining of DNA with ethidium bromide (FIG. 2). The use of a moresensitive staining method conclusively indicates that the plasmid DNAcould be partitioned to the top phase in the primary system and thewater phase in thermoseparating system. No plasmid DNA is partitioned tothe bottom phase neither in the primary step nor in the thermoseparatingstep.

Partitioning of Total Protein

In the primary purification step in a process for plasmid isolation themain objective is to remove the dominating contaminants in the solutioncontaining the plasmid DNA. When the bacteria are lysed in the alkalinelysis step protein, RNA, genomic DNA, cells and cell debris arereleased. After the neutralisation step in the alkaline lysis the lysateis centrifuged and most of these contaminants are removed but there willstill be significant contaminants present in the sample. Analysis of thetotal protein partitioning in the first step showed a K value of 1.4.This gives a 65% yield of total protein in the top phase. The system candiscard 35% of the proteins in the first step. In the thermoseparationstep the yield of the protein is 100%. Thus, the system can discardproteins to an extent of 35%.

Concentration of the Plasmid DNA

When a primary purification step is designed one of the issues are todecrease the process volume of the working solution. In a two-phasesystem this can be achieved by decreasing the volume of the top-phase.By moving along a tie-line in a phase diagram the volume ratio betweenthe two phases can be altered without changing the partitioning of thesubstance. If the dextran concentration is increased in the bottom phasea larger volume of the bottom phase is created and thus decreasing thevolume of the top phase. Four different systems composed of 4.5% (w/w)EO₅PO₅0/4.5% (w/w) Dextran T 500, 4% (w/w) EO₅₀PO₅₀/6% (w/w) Dextran T500, 3% (w/w) EO₅₀PO₅₀/7% (w/w) Dextran T 500, 3% (w/w) EO₅₀PO₅₀/8%(w/w) Dextran T 500 and 2.5% (w/w) EO₅₀PO₅₀/9% (w/w) Dextran T 500 werestudied. Analysis of the systems on agarose gel (FIG. 4.) shows that theextreme partitioning of plasmid DNA can still be achieved if the volumeis decreased in the top-phase. In the system comprising of 2.5% (w/w)EO₅₀PO₅₀/9% (w/w) Dextran T 500 a volume ratio (V_(T)/V_(B)) between thetop and bottom phase was 0.24. This leads to a concentration in the topphase of the isolated plasmid by a factor of 3 relative the alkalinelysis solution. In all of these systems RNA is partly discarded to thebottom phase.

Analysis of Plasmid DNA on Group Separation Chromatography

Analysis of plasmid DNA was performed on group separationchromatography. Separation between all forms of plasmid DNA and RNA canbe achieved. Results show that the total yield of the plasmid in the topwater phase after thermoseparation compared to the starting material is103.3%. The yield of RNA in the same sample according to chromatographyresults is 27%. This means that the aqueous two-phase system is able toremove 63% of the contaminating RNA.

Example 2 Separation of Plasmid pJV4 from an E. coli Lysate, Desaltingby Diafiltration

A total amount of 75 g cell paste from TG1/pJV4 cells were lysed andfurther treated as described in Example 1 above. The final lysate volumewas 2 L after final preparations.

Diafiltration

A polysulfon hollow fibre cartridge (A/G Technology Corporation,Needham, Mass., USA) with a 100 000 Da cut off was used fordiafiltration (Lot no. 96992057061, Lumen id: 0.5 mm, area 650 m²).Before use, the cartridge was washed with MilliQ water to removecontaminants of glycerol. The system was recirculated with 5 l of MilliQwater for 10 min and than with fresh 5 mM of Na-phosphate buffer for 10min. A volume of 350 ml clarified lysate was diafiltrated towards a 5 mMNa-phosphate buffer pH 7.0. For complete buffer exchange, four samplevolumes of 5 mM Na-phosphate buffer was used. Final retentate volumeafter diafiltration was 300 ml, which gave a volume reduction from 350ml to 300 ml. After diafiltration the column was washed with 100 ml 5 mMbuffer to remove bound plasmid DNA from the column.

A second diafiltration was performed since four buffer volumes of bufferwere not enough for complete buffer exchange. The diafiltrated lysatewas diafiltrated once more towards a 5 mM NaP buffer, pH 7. Another 8buffer volumes were used to achieve complete buffer exchange. The finaldiafiltrated sample was further extracted in an aqueous two-phasesystem.

Aqueous Two-Phase Systems

The systems were prepared as described in Example 1 above.

Results Example 2 Partitioning of Diafiltrated Plasmid DNA in aThermoseparating System

The diafiltrated lysate was partitioned in an aqueous two-phase systemcomposed of 4.5% (w/w) EO₅₀PO₅₀, 4.5% (w/w) Dextran T 500 and 50 mMNa₂HPO₄. All chemicals and the diafiltrated lysate was added to a glasstube to a final weigtht of 10 g. The system was then separated bycentrifugation (1600 g, 10 min) at room temperature. The phases wereseparated in one top phase and one bottom phase and isolated in newcontainers.

The top phase was put in a water bath at 55° C. The system phaseseparated into two new phases, one water phase and one dense polymerphase. The thermoseparating step was utilised for separating the targetplasmid from the EO₅₀PO₅₀ polymer, and isolating the plasmid in a waterphase with low (<1%) polymer solution (FIG. 1). The phases from theaqueous two-phase extraction were analysed with agarose gelelectrophoresis and size exclusion chromatography (group separation).The results from the group separation showed a one sided partitioning ofthe plasmid to the top phase (Table 1). The calculations of theconcentration of the plasmid DNA are performed from a standard curve.From the agarose gel electrophoresis (FIG. 5.) it can be seen in lane 4that no plasmid is partitioned to the bottom phase in the firstextraction step while a lot of the RNA is discarded. Thus, a desaltingof the alkaline lysate with diafiltration can be achieved followed byextraction in an aqueous two-phase system. A yield of 100% in thethermoseparated top phase can be achieved.

TABLE 1 Results from the group separation chromatography on adiafiltrated lysate and the phases from the extraction from an aqueoustwo-phase system. Amount A 260 Conc pDNA Vol. Rec. Sample mAu Dilution(μg/ml) (mg) (ml) (%) Diafiltrated ly- 1163.9 2 160.6 1.152 7.175 100 sate (Start ma- terial in ATPS) Thermosep- 1743.5 2 240.6 1.126 4.68   97.8* arated top phase 1 Thermosep- 1835.6 2 253.3 1.185 4.68 103*arated top phase 2 # Conc factor is calculated as V starting lysate(ml)/V Diafiltrated lysate (ml) *Recovery calculated as amount pDNA inthermoseparated top phase/amount pDNA in lysate. Double samples.

Example 3 Separation of Plasmid pJV4 from an E. coli Lysate, Desaltingby Ultrafiltration

A total amount of 75 g cell paste from TG1/pJV4 cells were lysed andfurther treated as described in Example 1 above. The final lysate volumewas 2 L after final preparations.

Ultrafiltration

The same cartridge with same cut off as earlier described fordiafiltration was also used for ultrafiltration. A volume of 1000 ml ofclarified lysate was ultrafiltrated until a volume of 225 ml wasreached. The ultrafiltrated lysate was than dialysed against 800 ml of 5mM NaP buffer. The lysate was dialysed until the buffer was finished.The lysate was than ultrafiltrated again until a final volume of 72 mland further extracted in an aqueous two-phase system.

Results Example 3 Partitioning of Ultrafiltrated Plasmid DNA in aThermoseparating System

The ultrafiltrated lysate was partitioned in an aqueous two-phase systemas described earlier. From the agarose gel electrophoresis (FIG. 6.) itcan be seen that an almost one sided partition was achieved of theplasmid. A small loss of the plasmid to the bottom phase is achieved inthis system. This is probably due to the high plasmid concentration inthe system e.g. 2 mg/ml or another possibility could be insufficientbuffer exchange. The results of samples from ultrafiltration andtwo-phase extraction are presented in table 2. The ultrafiltrated lysateis concentrated 13.9 times from the starting material. No precipitationof RNA and DNA was visible in the ultrafiltrated lysate. Both a bufferexchange and a concentration of the lysate can be achieved withultrafiltration. The ultrafiltrated lysate was compatible with theaqueous two-phase system but with some loss of the plasmid DNA to thebottom phase depending either on capacity limitations of the aqueoustwo-phase system or incomplete buffer exchange. The yield of plasmid DNAin the thermoseparated top phase was 85%.

TABLE 2 Results from group separation chromatography for anultrafiltrated lysate and a thermoseparated top-phase from two-phaseextraction. Amount A 260 Conc pDNA Vol. Rec. Sample mAu Dilution (μg/ml)(mg) (ml) (%) Ultrafiltrated 514.117 50 1773.7 12.73 7.175 100 lysate(Start material in ATPS) Thermo- 692.7 50 2389.8 10.75 4.5*  85*separated top phase # Conc factor is calculated as V starting lysate(ml)/V Diafiltrated lysate (ml) *Recovery calculated as amount pDNA inthermoseparated top phase/amount pDNA in lysate

1. A method for the purification of plasmid DNA in an aqueous two-phasesystem, comprising: (a) providing a composition including a firstpolymer EO₅₀PO₅₀, a second polymer Dextran T 500 and, optionally, asalt; (b) contacting said composition with an aqueous solutioncomprising plasmid DNA and RNA to form a mixture which contains fromabout 4.5% (w/w) EO₅₀PO₅₀/4.5%(w/w) Dextran T 500, to about2.5%(w/w)EO₅₀PO₅₀/9% (w/w) Dextran T 500; (c) providing a phaseseparation wherein plasmid DNA is partitioned to a top aqueous phasewhile RNA partitions predominantly to a lower phase, and subsequentlyisolating the top aqueous phase; (d) increasing the temperature of theisolated top aqueous phase to a temperature above the cloud point of thefirst polymer and below a temperature where plasmid DNA is degraded andsubsequently isolating a top aqueous phase so formed; and, optionally,(e) performing a chromatography step to recover the plasmid DNA from theisolated top phase of step (d).
 2. The method of claim 1, wherein theamount of the first polymer is about 4.5%(w/w) and the amount of thesecond polymer is about 4.5%(w/w) of the mixture in step (b).
 3. Themethod of claim 1, wherein the aqueous solution that includes plasmidDNA is a cell lysate, and wherein said method further comprises a stepfor desalting the cell lysate before step (b).
 4. The method of claim 1,wherein the contacting according to step (b) involves mixing at roomtemperature.
 5. The method of claim 1, wherein the isolation accordingto step (c) and/or step (d) is by centrifugation.
 6. The method of claim1, wherein the salt concentration in said composition in step (a) is atleast ten times above that of the aqueous solution.
 7. The method ofclaim 2, wherein the salt concentration in said composition in step (a)is at least ten times above that of the aqueous solution.